Assisted reproductive technologies such as artificial insemination have been practiced commercially with livestock and horses for almost a century, and with poultry, humans and other species for almost a half century. Artificial insemination requires a sample of semen consisting of spermatozoa produced by the testes and seminal plasma contributed by epithelial cells of the epididymides, deferent ducts and accessory sex glands. In addition to water, seminal plasma contains numerous proteins and glycoproteins, phospholipids, lipids, sugars and other carbohydrates, and ions.
It was early recognized that those portions of seminal plasma originating from the deferent ducts and accessory sex glands are not essential for spermatozoa to acquire or to retain fertilizing capability. Furthermore, for some species, or for certain males of a species, retention of trace or moderate amounts of seminal plasma in association with the sperm is desirable for retention of fertilizing capability during storage, whereas for others seminal plasma is deleterious. The beneficial or deleterious effects of seminal plasma on sperm became increasingly evident as procedures were developed to store sperm for several hours or days at 4 to 22° C., or for years at −196° C.
It has become evident that certain proteins or glycoproteins, phospholipids, and other components of seminal plasma can be loosely bound to sperm, presumably by interaction with the glycocalyx surrounding the sperm plasma membrane, or less frequently by true incorporation into the plasma membrane. Such molecules might be involved in binding of sperm to eggs. It has been reported that a crude mixture of proteins, extracted from human sperm by treatment with 0.6M KCl, increased the number of human sperm bound to a human zona pellucida in vitro. Jean et al., “Increased Zona-binding Ability After Incubation of Spermatozoa with Proteins Extracted from Spermatozoa of Fertile Semen”, J. of Reproduction and Fertility (1995), Vol. 105, pp. 43-48. However, in contrast to the present invention, that protein was not obtained by freezing sperm, and that publication gives no clue to the identity of the active molecule(s), mechanism of action, or species specificity.
The initial event in the fertilization process is binding of one or more sperm to the egg investments. Most research has focused on the molecular nature of the egg coverings in mammals (zona pellucida, an acellular coating outside a mammalian oocyte, and plasma membrane) and the nature of enzymes or glycoproteins on or in the plasma membrane or acrosome of sperm in mammals and invertebrates. Little research has been done on the molecular nature of avian sperm-egg binding, especially as it relates to the “sperm side” of the interaction.
In mammals, a spermatozoon is considered to bind to the oocyte through a series of egg-binding proteins (ligands) located on the surface of the spermatozoon which interact with appropriate members of a series of sperm receptors located on the investments of the oocyte, namely the zona pellucida and oocyte plasma membrane.
Sperm-egg binding proteins generally are considered to be transmembrane proteins or glycoproteins, possibly with enzymatic activity, with an extracellular domain that interacts with a specific sperm receptor. The consensus scenario which has evolved is one of sequential binding: (a) loose binding to the zona pellucida via one or more molecules located on the sperm plasma membrane, (b) tight binding to the zona pellucida via (a) molecule(s) located on the plasma membrane and/or the acrosomal membrane, and (c) tight binding to the oocyte plasma membrane followed by fusion of the sperm and egg plasma membranes and entrance of the entire spermatozoon into the oocyte. Thus, species-specific adhesion between sperm and eggs is attributable to complexes formed between egg-binding proteins on sperm and complementary sperm receptors on eggs. Species non-specific egg-binding proteins are thought to exist, because binding to zona-free hamster oocytes occurs with sperm of many species, but at least one species-specific egg-binding protein also must be located on the sperm surface. To date, no universal class of candidate molecules has been identified as the species-non-specific, loose egg-binding component.